Dfc400

To investigate the route of water intake in ticks, 1.28 µL of 1 mM rhodamine123 in water (Sigma-Aldrich, St. Louis, MO, USA) was filled in a microcapillary tube (Diameter ∼0.11 mm, Sigma-Aldrich, St. Louis, MO, USA) and the tube was placed onto the mouthparts of dehydrated unfed female ticks. Ticks were allowed to ingest Rh123 under rehydration conditions (RH 98%) at room temperature for 30 min. After ingestion, we quantified the ingested amount and traced the locations of fluorescence signal in tick organs, specifically in the salivary glands and the gut diverticular. The volume ingested was calculated by the equation for cylinder volume (V = πr²h). For calculation of volume, the reduced length (h) of fluid in microcapillary tubes was measured under a grid microscope and analyzed with r² = 0.0127. The locations of fluorescence in the internal organs were identified after dorsal integument of ticks was removed by a surgical scalpel. Images were captured using a camera (DFC400) attached to a stereo microscope (M205FA; Leica, Heerbrugg, Switzerland). Salivary glands were subsequently dissected out, fixed in 4% paraformaldehyde at room temperature for 30 min, washed in PBST (0.1% Triton X-100), and imaged on a confocal microscope (LSM700; Zeiss, Oberkochen, Germany).

RNA sense and anti-sense probes were synthesized from pcsDest2-gdf6a and pcsDest2-gdf6b constructs using DIG RNA Labeling Kit (Roche) per the manufacturer’s instruction. Wild-type embryos of the appropriate stage were fixed in 4% PFA at 4°C for 24 hr. Following fixation, embryos were dehydrated in methanol at stored at −20°C. Whole mount in situ hybridization was performed as previously described (Reichert et al., 2013 (link)). Hybridized probes were detected using anti-digoxigenin (DIG) antibodies tagged with alkaline-phosphatase (AP) (Roche) using NBT/BCIP (Roche) solution per the manufacturer’s instructions. Stained embryos were mounted in 2.5% methylcellulose and imaged using a Leica M165FC microscope and Leica DFC400 camera. Specificity of the probes was verified using sense probes synthesized from the same construct.

After fluoride water treatment with control-chow or 4PBA-chow for 6 weeks, animals were euthanized. Heads were cleaned of fur and skin. Photographs of the maxillary and mandibular incisors were taken using a Nikon SMZ745T microscope and Leica DFC400 digital camera under standard white balance and lighting conditions.

Images were acquired with an inverted microscope (Leica-DMI6000B) equipped with Leica DFC365FX and DFC400 cameras and ×10 and ×20 magnification objectives. The Leica Application Suite software was used for acquisition, while Photoshop was used to generate merged images.

Images were acquired with an inverted microscope (Leica-DMI6000B) equipped with Leica DFC365FX and DFC400 cameras and 20× and 40× magnification objectives. Necrotic myofibers were defined as pink pale patchy fibers, and phagocyted myofibers were defined as pink pale fibers invaded by basophilic single cells (MPs). For the quantification of CSA, analyses were done on damaged TA, which presented at least 75% of injured muscle. At least 8 pictures in different fields were taken and at least 500 myofibers were analyzed. For each condition of each experiment, at least 8 fields chosen randomly were counted. The number of labelled MPs or mpcs was calculated using the cell tracker in ImageJ software and expressed as a percentage of total MPs or mpcs. Fusion index was the number of nuclei within myotubes divided by the total number of nuclei.